6-Aryl-7-halo-imidazo[1,2-a]pyrimidines as anticancer agents

ABSTRACT

This invention relates to certain 6-aryl-7-halo-imidazo[1,2-a]pyrimidines or pharmaceutically acceptable salts thereof, and compositions containing said compounds or pharmaceutically acceptable salts thereof, wherein said compounds are anti-cancer agents useful for the treatment of cancer in mammals by promotion of microtubule polymerization. This invention further relates to a method of treating or inhibiting the growth of cancerous tumor cells and associated diseases in a mammal and further provides a method for the treatment or prevention of cancerous tumors that express multiple drug resistance (MDR) or are resistant because of MDR, in a mammal in need thereof which method comprises administering to said mammal an effective amount of said compounds or pharmaceutically acceptable salts thereof.

“This application claims priority from copending provisional ApplicationNo. 60/505,486 filed Sep. 24, 2003 the entire disclosure of which ishereby incorporated by reference”

FIELD OF THE INVENTION

The present invention relates to 6-aryl-7-halo-imidazo[1,2-a]pyrimidinecompounds or pharmaceutically acceptable salts thereof, and compositionscontaining said compounds wherein said compounds are anti-cancer agentsuseful for the treatment of cancer in mammals. Compounds of theinvention are useful for the treatment or prevention of cancerous tumorsthat express multiple drug resistance (MDR) or are resistant because ofMDR. Further, compounds of the invention are useful for treating orinhibiting the growth of cancerous tumor cells and associated diseasesin a mammal in need thereof by promotion of microtubule polymerization.

BACKGROUND OF THE INVENTION

Most of the cytostatics in use today either inhibit the formation ofessential precursors for biosynthesis of DNA or block DNA polymerases orinterfere with the template function of DNA because DNA was the primarytarget for developing therapeutic drugs for chemotherapy. Unfortunately,inhibition of the formation of essential precursors for biosynthesis ofDNA or blocking DNA polymerases or interference with the templatefunction of DNA also affects normal tissues.

Antimicrotubule drugs are a major category of anticancer agents(Rowinsky, E. K., and Tolcher, A. W. Antimicrotubule agents. In: V. T.Devita, Jr., S. Hellman, and S. A. Rosenberg (eds.), Cancer Principlesand Practice, Ed. 6, pp. 431-452. Philadelphia: Lippincott Williams andWilkins, 2001). They work by interfering with the function of cellularmicrotubules, particularly the mitotic spindle. The disruption of normalspindle function leads to apoptotic cell death.

Currently, there are three major classes of known antimicrotubulepharmacological agents. Each has a distinct binding region on β-tubulinand distinct effects on microtubule function. These classes are: 1)taxane-site agents which promote microtubule formation and stabilizemicrotubules; 2) vinca/peptide-site agents which destabilizemicrotubules and often induce formation of abnormal polymers oraggregates at high concentrations; and 3) colchicine-site agents whichalso destabilize microtubules and generally do not induce other polymers(Hamel, E. Antimitotic natural products and their interactions withtubulin. Med. Res. Rev., 16: 207-231, 1996). Most of the ligands for allthree classes of sites are natural products or semi-syntheticderivatives of natural products.

Paclitaxel and its semisynthetic derivative docetaxel (Taxotere®)interfere with microtubule formation and stabilize microtubules.Paclitaxel (Taxol®), is a diterpene isolated from the bark of theWestern (Pacific) yew, Taxus brevifolia and is representative of a newclass of therapeutic agent having a taxane ring system. It wasadditionally found in other members of the Taxacae family including theyew of Canada (Taxus canadensis) found in Gaspesia, eastern Canada andTaxus baccata found in Europe whose needles contain paclitaxel andanalogs and hence provide a renewable source of paclitaxel andderivatives. The crude extract was tested for the first time during the1960s and its active principle was isolated in 1971 and the chemicalstructure identified (M. C. Wani et al, J. Am. Chem. Soc., 93, 2325(1971)). Further, a wide range of activity over melanoma cells,leukemia, various carcinomas, sarcomas and non-Hodgkin lymphomas as wellas a number of solid tumors in animals was shown through additionaltesting. Paclitaxel and its analogs have been produced by partialsynthesis from 10-deacetylbaccatin III, a precursor obtained from yewneedles and twigs, and by total synthesis (Holton, et al., J. Am. Chem.Soc. 116:1597-1601 (1994) and Nicolaou, et al., Nature 367:630-634(1994)). Paclitaxel has been demonstrated to possess antineoplasticactivity. More recently, it was shown that the antitumor activity ofpaclitaxel is due to a promotion of microtubule polymerization (Kumar,N., J. Biol. Chem. 256:10435-10441 (1981); Rowinsky, et al., J. Natl.Cancer Inst., 82:1247-1259 (1990); and Schiff, et al., Nature,277:665-667 (1979)). Paclitaxel has now demonstrated efficacy in severalhuman tumors in clinical trials (McGuire, et al., Ann. Int. Med.,111:273-279 (1989); Holmes, et al., J. Natl. Cancer Inst., 83:1797-1805(1991); Kohn et al., J. Natl. Cancer Inst., 86:18-24 (1994); and A.Bicker et al., Anti-Cancer Drugs, 4, 141-148 (1993).

Two taxane-site agents (paclitaxel and docetaxel) and threevinca/peptide-site agents (vinblastine, vincristine, and vinorelbine)are used clinically to treat various human cancers. Taxanes have provento be of greater utility against solid tumors (e.g., lung, breast,ovarian) than the vinca alkaloids, suggesting that agents that promotemicrotubule formation might be superior clinically to those thatdestabilize microtubules. Colchicine-site agents are not usedtherapeutically.

Despite the widespread clinical use of paclitaxel and docetaxel, thesedrugs have several limitations that create a need for improved agents.First, many tumors are inherently resistant (e.g., colon tumors) orbecome resistant after multiple cycles of treatment, at least in partdue to the expression of drug transporters located in cancer cellmembranes that pump the drugs out of cells and thereby decrease theirefficacy (Gottesman, M. M. Mechanisms of cancer drug resistance. Annu.Rev. Med., 53: 615-627, 2002). The best known of these transporters isP-glycoprotein. Accordingly, there is a need for new agents withtaxane-like effects on microtubule polymerization that are notsubstrates of P-glycoprotein or other such pumps and that therefore willovercome this cause of taxane resistance in patients.

Second, paclitaxel and docetaxel have poor water solubility andpaclitaxel must be formulated in Cremophor EL, a vehicle that inducesserious hypersensitivity reactions (Li, C. L., Newman, R. A., andWallace, S. Reformulating paclitaxel. Science & Medicine,January/February: 38-47, 1999). Patients are typically premedicated withcorticosteroids and antihistamines before administration of paclitaxelto minimize these toxicities. Accordingly, there is a need for newagents with taxane-like effects on microtubule polymerization that arehighly water soluble and can be administered in physiological saline orother suitable non-toxic vehicle.

Third, paclitaxel is a natural product having a highly complexstructure, and docetaxel is a closely related semisynthetic derivative.Therefore there is a need for compounds which are readily availablethrough synthesis, are structurally different from taxanes and whichhave taxane-like effects on microtubule polymerization.

Accordingly, there is still a need in the art for cytotoxic agents foruse in cancer therapy. In particular, there is a need for cytotoxicagents which inhibit or treat the growth of tumors which have an effectsimilar to paclitaxel and interfere with the process of microtubuleformation. Additionally, there is a need in the art for agents whichaccelerate tubulin polymerization and stabilize the assembledmicrotubules.

Further, it would be advantageous to provide new compounds which providea method of treating or inhibiting cell proliferation, neoplastic growthand malignant tumor growth in mammals by administering compounds whichhave paclitaxel like anticancer activity.

Additionally, it would be advantageous to provide new compounds whichprovide a method for treating or inhibiting growth of cancerous tumorsthat express multiple drug resistance (MDR) or are resistant because ofMDR.

Further, it would be advantageous to provide new compounds which providea method of treating or inhibiting the growth of cancerous tumors in amammal with inherent or acquired resistance to chemotherapeutic agentsand in particular antimitotic agents.

Described in the art is the preparation and use of substitutedtriazolopyrimidines in agriculture as fungicides and are disclosed inU.S. Pat. Nos. 5,593,996; 5,756,509; 5,948,783; 5,981,534; 5,612,345;5,994,360; 6,020,338; 5,985,883; 5,854,252; 5,808,066; 5,817,663;5,955,252; 5,965,561; 5,986,135; 5,750,766; 6,117,865; 6,117,876;6,124,301; 6,204,269; 6,255,309; 6,268,371; 6,277,856; 6,284,762;6,297,251; 6,387,848; US Patent Application PublicationUS2002/0045631A1; US2002/0061882A1; US20030055069A1 and InternationalPublication Numbers: WO98/46607; WO98/46608; WO99/48893; WO99/41255;WO00/18227; WO01/35738A2; WO02/46195A1; WO02/067679A1; WO02/083676A1;EPO 834513A2; EPO 782997A2; EPO550113B1; FR2784381A1; EPO 989130A1;WO98/41496; WO94/20501; EPO 945453A1; EPO 562615A1; EPO 562615B1; EP 0550113A2; EP 0 943241 B1; EP 0 988790 B1 and having the followinggeneral formula:

Also known are the use of triazolopyrimidines as anticancer agentshaving the structural formula

described in WO02/02563 A2.

5,7-Dihydroxyimidazo[1,2-a]pyrimidine without phenyl substitution at the6-position is known (R. P. Rao et al, J. Het. Chem. 1021 (1973)). Alsoknown is 5,7-dichloroimidazo[1,2-a]pyrimidine without phenylsubstitution at the 6-position (G. R. Revankar, et al, J. Med. Chem. 18,1253 (1975)).

EP 0,770,615 provides a process for the synthesis ofdihaloazolopyrimidines of the formula

wherein:X₁ is chlorine or bromine;R is optionally substituted phenyl;X, Y, and Z are CR₁ or N and further described is the synthesis of5,7-dihydroxy-6-(2-chloro-6-fluorophenyl)benzimidazopyridine having thestructural formula

Described in JP2001043978 are diazaindolizines represented by thegeneric structure

wherein said compounds are useful as electroluminescent elements.

Described in WO 03/022850 A1 are imidazo[1,2-a]pyrimidines representedby the following general formula

wherein said compounds are useful as fungicides.

The compounds of this invention are a new class of taxane-like agentsthat satisfy the hereinbefore described needs, and that differ insignificant ways from the previously known classes of antimicrotubulecompounds. The compounds of this invention bind at the vinca site ofβ-tubulin, yet they have many properties that are similar to taxanes anddistinct from vinca-site agents. In particular, the compounds of thisinvention enhance the polymerization of microtubule-associated protein(MAP)-rich tubulin in the presence of GTP at low compound:tubulin molarratios, in a manner similar to paclitaxel and docetaxel. Representativeexamples of the compounds of this invention also induce polymerizationof highly purified tubulin in the absence of GTP under suitableexperimental conditions, an activity that is a hallmark of taxanes. Thecompounds of this invention are potently cytotoxic for many human cancercell lines in culture, including lines that overexpress the membranetransporters MDR (P-glycoprotein), MRP, and MXR, thus making them activeagainst cell lines that are resistant to paclitaxel and vincristine.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided compoundsrepresented by Formula (I):

wherein:R¹ is selected from

and C₆-C₈ cycloalkyl;R² is a moiety of the formula

R³ is H, or C₁-C₃ alkyl;R⁴ is H, or C₁-C₃ alkyl; orR³ and R⁴ when optionally taken together form a 6 to 8 memberedsaturated heterocyclic ring having 1-2 nitrogen atoms, 0-1 oxygen atomsand 0-1 sulfur atoms, and optionally substituted with C₁-C₃ alkyl;R⁵ is H, C₁-C₃ alkyl or C₁-C₃ fluoroalkyl;Y is a moiety of the formula —O(CH₂)_(n)Q;n is an integer of 2, 3 or 4;

Q is —OH, or —NR⁶R⁷;

R⁶ and R⁷ are independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁶ is C₁-C₃ alkyl;L¹, L², L³, and L⁴ are each independently H, F, Cl, Br or CF₃;

X is Cl or Br;

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the invention are compounds according toFormula (Ia):

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the invention are compounds according toFormula (Ib):

or pharmaceutically acceptable salts thereof.

A further preferred embodiment of the present invention providescompounds according to Formula (I) or pharmaceutically acceptable saltsthereof wherein R² is a moiety of the formula

Among the more preferred group of compounds of this invention accordingto Formula (Ia) including pharmaceutically acceptable salts thereof arethose wherein:

R² is

n is 3;

X is Cl or Br;

Y is a moiety of the formula —(CH₂)_(n)Q;R³ is H or methyl;

R⁴ is H; Q is —NR⁶R⁷; R⁵ is CF₃;

R⁶ and R⁷ are each independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;

L¹ and L⁴ are F; L² and L³ are H;

or pharmaceutically acceptable salts thereof.Among the more preferred group of compounds of this invention accordingto Formula (Ib) including pharmaceutically acceptable salts thereof arethose selected from the subgroups a) and b) below:a)

R² is

n is 3;

X is Cl or Br;

Y is a moiety of the formula —(CH₂)_(n)Q;R³ is H or methyl;

R⁴ is H; Q is —NR⁶R⁷; R⁵ is CF₃;

R⁶ and R⁷ are each independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;

L¹ and L⁴ are F; L² and L³ are H;

or pharmaceutically acceptable salts thereof andb)

R² is

n is 3;

X is Cl;

Y is a moiety of the formula O(CH₂)_(n)Q;

Q is —NR⁶R⁷; R⁴ is H;

R⁶ is methyl;R⁷ is H or methyl;

L¹ and L⁴ are F; L² and L³ are H;

or pharmaceutically acceptable salts thereof.

Preferred compounds of this invention according to Formula (I) includingpharmaceutically acceptable salts thereof are those wherein R¹ is C₆-C₈cycloalkyl.

Among the more preferred group of compounds of this invention accordingto Formula (I) including pharmaceutically acceptable salts thereof arethe subgroup below:

R¹ is C₆-C₈ cycloalkyl;

R² is

n is 3;

X is Cl;

Y is a moiety of the formula —O(CH₂)_(n)Q;

Q is —NR⁶R⁷;

R⁶ is methyl;R⁷ is H or methyl;

L¹ and L⁴ are F; L² and L³ are H;

or pharmaceutically acceptable salts thereof.

Among the most preferred group of compounds of this invention accordingto Formula (Ia) including pharmaceutically acceptable salts thereof arethose of the group below:

R² is

X is Cl;

n is 3;Y is O(CH₂)_(n)Q;

Q is —NR⁶R⁷;

R³ is H or methyl;

R⁴ is H; R⁵ is CF₃;

R⁶ is methyl;R⁷ is H or methyl;

L¹ and L⁴ are F; L² and L³ are H;

or pharmaceutically acceptable salts thereof.

DEFINITIONS

As utilized herein, the term “alkyl”, alone or in combination, means astraight-chain or branched-chain alkyl radical containing from 1 to 3,preferably from 1 to 2, carbon atoms. Examples of such radicals includemethyl, ethyl, n-propyl, isopropyl, and the like.

Fluoroalkyl means an alkyl group up to 3 carbon atoms wherein eachhydrogen may be independently replaced by a fluorine atom.

The term alkali metal hydride includes lithium, potassium or sodiumhydride.

The term alkali metal hydroxide includes lithium, potassium or sodiumhydroxide.

The term alkali metal carbonate includes lithium, potassium or sodiumcarbonate.

Phenyl as used herein refers to a 6-membered carbon aromatic ring.

Cycloalkyl as used herein means a saturated carbocyclic monocyclic ringhaving from 6 to 8 carbon atoms optionally substituted with C₁-C₃ alkyl.Non-limiting representative examples include: cyclohexyl, cycloheptyland cyclooctyl.

As used herein a saturated heterocyclic ring is a 4 to 6 membered ringwith 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atomsoptionally substituted with C₁-C₃ alkyl. Non-limiting representativeexamples include: morpholine, piperidine, pyrrolidine, piperazine, andazetidine.

The term t-BOC as used herein means tert-butoxy carbonyl.

Included in the scope of the present invention are (R) and (S) isomersof compounds of Formula (I) having a chiral center and the racematesthereof.

The present invention provides a method of treating or inhibiting thegrowth of cancerous tumor cells and associated diseases in a mammal byadministering an effective amount of the compounds of Formula (I) andpharmaceutically acceptable salts thereof in need thereof.

The present invention also provides a method of treating or inhibitingthe growth of cancerous tumor cells and associated diseases in mammalsin need thereof by interacting with tubulin and microtubules bypromotion of microtubule polymerization which comprises administering tosaid mammal an effective amount of the compounds of Formula (I) andpharmaceutically acceptable salts thereof.

The present invention further provides method for the treatment orprevention of tumors that express multiple drug resistance (MDR) or areresistant because of MDR in a mammal in need thereof which methodcomprises administering to said mammal an effective amount of suchcompounds or pharmaceutically acceptable salts thereof.

This invention also provides a method of promoting tubulinpolymerization in a tubulin containing system by contacting said tubulincontaining system with an effective amount of a compound of Formula (I)or pharmaceutically acceptable salts thereof.

Additionally this invention provides a method of stabilizingmicrotubules in a tubulin containing system which comprises contactingsaid tubulin containing system with an effective amount of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof.

Additionally this invention provides a method of treating, inhibitingthe growth of, or eradicating a tumor in a mammal in need thereofwherein said tumor is resistant to at least one chemotherapeutic agentwhich comprises administering to said mammal an effective amount of thecompounds of Formula (I) and pharmaceutically acceptable salts thereof.

Further this invention provides a compound of Formula (I) in combinationor association with a pharmaceutically acceptable carrier. Inparticular, the present invention provides a pharmaceutical compositionwhich comprises a compound of Formula (I) and a pharmaceuticallyacceptable carrier.

Additionally this invention provides a method of treating, inhibitingthe growth of, or eradicating a tumor in a mammal in need thereofwherein said tumor is resistant to at least one chemotherapeutic agentwhich comprises administering to said mammal an effective amount of thecompounds of Formula (I) and pharmaceutically acceptable salts thereof.

The compounds of this invention may contain an asymmetric carbon atomand some of the compounds of this invention may contain one or moreasymmetric centers and may thus give rise to stereoisomers, such asenantiomers and diastereomers. The stereoisomers of the instantinvention are named according to the Cahn-Ingold-Prelog System. Whileshown without respect to stereochemistry in Formula (I), the presentinvention includes all the individual possible stereoisomers; as well asthe racemic mixtures and other mixtures of R and S stereoisomers(scalemic mixtures which are mixtures of unequal amounts of enantiomers)and pharmaceutically acceptable salts thereof. Included in the scope ofthe present invention are (R) and (S) isomers of compounds of generalFormula (I) having a chiral center and the racemates thereof. Thepresent invention encompasses all stereoisomers of the compounds whetherfree from other stereoisomers or admixed with other stereoisomers in anyproportion and thus includes, for instance, racemic mixture ofenantiomers as well as the diastereomeric mixture of isomers. Theabsolute configuration of any compound may be determined by conventionalX-ray crystallography.

Optical isomers may be obtained in pure form by standard separationtechniques or enantiomer specific synthesis.

Particularly preferred are isomers of Formula (I) wherein R¹ is themoiety

having the (S) configuration.

Specifically preferred compounds of this invention according to Formula(I) are the following compounds or pharmaceutically acceptable saltsthereof selected from the group:

-   7-Chloro-6-[4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl]-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   3-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine,-   7-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   N-{3-[4-(7-chloro-5-cyclohexylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propyl}-N,N-dimethylamine,-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine.    Specifically preferred compounds of this invention according to    Formula (Ia) are the following compounds or pharmaceutically    acceptable salts thereof selected from the group:-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1S)-2,2,2-trifluoro-1-methyl    ethyl]imidazo[1,2-a]pyrimidin-5-amine.

Specifically preferred compounds of this invention according to Formula(Ib) are the following compounds or pharmaceutically acceptable saltsthereof selected from the group:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine

Also provided is a method of treating or inhibiting the growth ofcancerous tumor cells and associated diseases in a mammal in needthereof which comprises administering an effective amount of a compoundof Formula (II):

wherein:R¹ is selected from

and C₆-C₈ cycloalkyl;R² is a moiety of the formula

R³ is H, or C₁-C₃ alkyl;R⁴ is H, or C₁-C₃ alkyl; orR³ and R⁴ optionally taken together form a 6 to 8 membered saturatedheterocyclic ring having 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1sulfur atoms, and optionally substituted with C₁-C₃ alkyl;R⁵ is H, C₁-C₃ alkyl or C₁-C₃ fluoroalkyl;Y is H, F, Cl, or a moiety of the formula —O(CH₂)_(n)Q;n is an integer of 2, 3 or 4;

Q is —OH, or —NR⁶R⁷;

R⁶ and R⁷ are independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹, L², L³, and L⁴ each independently H, F, Cl, Br or CF₃;

X is Cl or Br;

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method oftreating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(IIa):

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method oftreating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(IIb):

or pharmaceutically acceptable salts thereof.

An additionally preferred embodiment of the present invention provides amethod of treating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(II) or pharmaceutically acceptable salts thereof wherein R¹ is C₆-C₈cycloalkyl.

A further preferred embodiment of the present invention provides amethod of treating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(II) or pharmaceutically acceptable salts thereof wherein R² is a moietyof the formula

A more preferred embodiment of the present invention provides a methodof treating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(II) including pharmaceutically acceptable salts thereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof treating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(IIa) including pharmaceutically acceptable salts thereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R³ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof treating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(II) including pharmaceutically acceptable salts thereof wherein:

R¹ is C₆-C₈ cycloalkyl;R² is the moiety

Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof treating or inhibiting the growth of cancerous tumor cells andassociated-diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound according to Formula(IIb) including pharmaceutically acceptable salts thereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A specific embodiment of the present invention provides a method oftreating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount a compound or pharmaceuticallyacceptable salts thereof, selected from the group:

-   5-Azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-5-piperidin-1-yl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   3-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine,-   7-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   N-{3-[4-(7-chloro-5-cyclohexylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propyl}-N,N-dimethylamine,-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method oftreating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound or pharmaceuticallyacceptable salts thereof according to Formula (IIa) selected from thegroup:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method oftreating or inhibiting the growth of cancerous tumor cells andassociated diseases in a mammal in need thereof which comprisesadministering an effective amount of a compound or pharmaceuticallyacceptable salts thereof according to Formula (IIb) selected from thegroup:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

Further provided is a method of promoting tubulin polymerization in atubulin containing system by contacting said tubulin containing systemwith an effective amount of a compound of formula (II) orpharmaceutically acceptable salts thereof wherein:

wherein:R¹ is selected from

and C₆-C₈ cycloalkyl;R² is a moiety of the formula

R³ is H, or C₁-C₃ alkyl;R⁴ is H, or C₁-C₃ alkyl; orR³ and R⁴ optionally taken together form a 6 to 8 membered saturatedheterocyclic ring having 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1sulfur atoms, and optionally substituted with C₁-C₃ alkyl;R⁵ is H, C₁-C₃ alkyl or C₁-C₃-fluoroalkyl;Y is H, F, Cl, or a moiety of the formula —O(CH₂)_(n)Q;n is an integer of 2, 3 or 4;

Q is —OH, or —NR⁶R⁷;

R⁹ and R⁷ are independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹, L², L³, and L⁴ each independently H, F, Cl, Br or CF₃;

X is Cl or Br;

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method ofpromoting tubulin polymerization in a tubulin containing system bycontacting said tubulin containing system with an effective amount of acompound of Formula (IIa):

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method ofpromoting tubulin polymerization in a tubulin containing system bycontacting said tubulin containing system with an effective amount of acompound of Formula (IIb):

or pharmaceutically acceptable salts thereof.

An additionally preferred embodiment of the present invention provides amethod of promoting tubulin polymerization in a tubulin containingsystem by contacting said tubulin containing system with an effectiveamount of a compound of Formula (II) or pharmaceutically acceptablesalts thereof wherein R¹ is C₆-C₈ cycloalkyl.

This invention provides a method of promoting tubulin polymerization ina tubulin containing system by contacting said tubulin containing systemwith an effective amount of a compound of formula (II) wherein R² is amoiety of the formula

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof promoting tubulin polymerization in a tubulin containing system withan effective amount of a compound of Formula (IIa) includingpharmaceutically acceptable salts thereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof promoting tubulin polymerization in a tubulin containing system bycontacting said tubulin containing system with an effective amount of acompound of Formula (II) including pharmaceutically acceptable saltsthereof are those wherein:

R¹ is C₆-C₈ cycloalkyl;R² is the moiety

Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof promoting tubulin polymerization in a tubulin containing system bycontacting said tubulin containing system with an effective amount of acompound of Formula (IIb) including pharmaceutically acceptable saltsthereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A specific embodiment of the present invention provides a method ofpromoting tubulin in a tubulin containing system by contacting saidtubulin containing system with an effective amount of a compound orpharmaceutically acceptable salts thereof according to Formula (II)selected from the group:

-   5-Azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-5-piperidin-1-yl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   3-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine,-   7-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   N-{3-[4-(7-chloro-5-cyclohexylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propyl}-N,N-dimethylamine,-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method ofpromoting tubulin polymerization in a tubulin containing system bycontacting said tubulin containing system with an effective amount of acompound or pharmaceutically acceptable salts thereof according toFormula (IIa) selected from the group:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method ofpromoting tubulin polymerization in a tubulin containing system bycontacting said tubulin containing system with an effective amount of acompound or pharmaceutically acceptable salts thereof according toFormula (IIb) selected from the group:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

The present invention further provides a method of stabilizingmicrotubules in a tubulin containing system which comprises contactingsaid tubulin containing system with an effective amount of a compound ofFormula (II):

wherein:R¹ is selected from

and C₆-C₈ cycloalkyl;R² is a moiety of the formula

R³ is H, or C₁-C₃ alkyl;R⁴ is H, or C₁-C₃ alkyl; orR³ and R⁴ optionally taken together form a 6 to 8 membered saturatedheterocyclic ring having 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1sulfur atoms, and optionally substituted with C₁-C₃ alkyl;R⁵ is H, C₁-C₃ alkyl or C₁-C₃ fluoroalkyl;Y is H, F, Cl, or a moiety of the formula —O(CH₂)_(n)Q;n is an integer of 2, 3 or 4;

Q is —OH, or —NR⁶R⁷;

R⁶ and R⁷ are independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹, L², L³, and L⁴ each independently H, F, Cl, Br or CF₃;

X is Cl or Br;

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method ofstabilizing microtubules in a tubulin containing system which comprisescontacting said tubulin containing system with an effective amount of acompound of Formula (IIa):

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method ofstabilizing microtubules in a tubulin containing system which comprisescontacting said tubulin containing system with an effective amount of acompound of Formula (IIb):

or pharmaceutically acceptable salts thereof.

An additionally preferred embodiment of the present invention provides amethod of stabilizing microtubules in a mammal in need thereof byadministering an effective amount of compounds of Formula (II) orpharmaceutically acceptable salts thereof wherein R¹ is C₆-C₈cycloalkyl.

A further preferred embodiment of the present invention provides amethod of stabilizing microtubules in a tubulin containing system whichcomprises contacting said tubulin containing system with an effectiveamount of a compound of Formula (II) or pharmaceutically acceptablesalts thereof wherein R² is a moiety of the formula

A more preferred embodiment of the present invention provides a methodof stabilizing microtubules in a tubulin containing system whichcomprises contacting said tubulin containing system with an effectiveamount of a compound of Formula (IIa) including pharmaceuticallyacceptable salts thereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof stabilizing microtubules in a tubulin containing system whichcomprises contacting said tubulin containing system with an effectiveamount of a compound of Formula (II) including pharmaceuticallyacceptable salts thereof wherein:

R¹ is C₆-C₈ cycloalkyl;R² is the moiety

Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodof stabilizing microtubules in a tubulin containing system whichcomprises contacting said tubulin containing system with an effectiveamount of a compound of Formula (IIb) including pharmaceuticallyacceptable salts thereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A specific embodiment of the present invention provides a method ofstabilizing microtubules in a tubulin containing system which comprisescontacting said tubulin containing system with an effective amount of acompound of Formula (II) or pharmaceutically acceptable salts thereofaccording to Formula (II) selected from the group:

-   5-Azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-5-piperidin-1-yl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   3-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine,-   7-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   N-{3-[4-(7-chloro-5-cyclohexylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propyl}-N,N-dimethylamine,-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method ofstabilizing microtubules in a tubulin containing system which comprisescontacting said tubulin containing system with an effective amount of acompound of Formula (IIa) or pharmaceutically acceptable salts thereofselected from the group:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method ofstabilizing microtubules in a tubulin containing system which comprisescontacting said tubulin containing system with an effective amount of acompound of Formula (IIb) or pharmaceutically acceptable salts thereofaccording to Formula (IIb) selected from the group:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

Also provided is a method for the treatment or prevention of tumors thatexpress multiple drug resistance (MDR) or are resistant because of MDRin a mammal in need thereof which method comprises administering to saidmammal an effective amount of a compound of formula (II):

wherein:R¹ is selected from

and C₆-C₈ cycloalkyl;R² is a moiety of the formula

R³ is H, or C₁-C₃ alkyl;R⁴ is H, or C₁-C₃ alkyl; orR³ and R⁴ when optionally taken together form a 6 to 8 memberedsaturated heterocyclic ring having 1-2 nitrogen atoms, 0-1 oxygen atomsand 0-1 sulfur atoms, and optionally substituted with C₁-C₃ alkyl;R⁵ is H, C₁-C₃ alkyl or C₁-C₃ fluoroalkyl;Y is H, F, Cl, or a moiety of the formula —O(CH₂)_(n)Q;n is an integer of 2, 3 or 4;

Q is —OH, or —NR⁶R⁷;

R⁶ and R⁷ are independently H or C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹, L², L³, and L⁴ each independently H, F, Cl, Br or CF₃;

X is Cl or Br;

or pharmaceutically acceptable salts thereof.A preferred embodiment of the present invention provides a method forthe treatment or prevention of tumors that express multiple drugresistance (MDR) or are resistant because of MDR in a mammal in needthereof which method comprises administering an effective amount of acompound of Formula (IIa):

or pharmaceutically acceptable salts thereof.

A preferred embodiment of the present invention provides a method forthe treatment or prevention of tumors that express multiple drugresistance (MDR) or are resistant because of MDR in a mammal in needthereof which method comprises administering an effective amount of acompound of Formula (IIb):

or pharmaceutically acceptable salts thereof.An additionally preferred embodiment of the present invention provides amethod for the treatment or prevention of tumors that express multipledrug resistance (MDR) or are resistant because of MDR in a mammal inneed thereof which method comprises administering an effective amount ofa compound of Formula (II) or pharmaceutically acceptable salts thereofwherein R¹ is C₆-C₈ cycloalkyl.

A further preferred embodiment of the present invention provides amethod for the treatment or prevention of tumors that express multipledrug resistance (MDR) or are resistant because of MDR in a mammal inneed thereof which method comprises administering an effective amount ofa compound of Formula (II) or pharmaceutically acceptable salts thereofwherein R² is a moiety of the formula

A more preferred embodiment of the present invention provides a methodfor the treatment or prevention of tumors that express multiple drugresistance (MDR) or are resistant because of MDR in a mammal in needthereof which method comprises administering an effective amount of acompound of Formula (IIa) including pharmaceutically acceptable saltsthereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodfor the treatment or prevention of tumors that express multiple drugresistance (MDR) or are resistant because of MDR in a mammal in needthereof which method comprises administering an effective amount of acompound of Formula (II) including pharmaceutically acceptable saltsthereof wherein:

R¹ is C₆-C₈ cycloalkyl;R² is the moiety

Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁵ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A more preferred embodiment of the present invention provides a methodfor the treatment or prevention of tumors that express multiple drugresistance (MDR) or are resistant because of MDR in a mammal in needthereof which comprises administering an effective amount of compoundsaccording to Formula (IIb) including pharmaceutically acceptable saltsthereof wherein:

R² is the moiety

R³ is H, or C₁-C₃ alkyl;

R⁴ is H;

R⁵ is C₁-C₃ fluoroalkyl;Y is F, or a moiety —O(CH₂)_(n)Q;n is 3;

Q is —NR⁶R⁷;

R⁶ and R⁷ are each independently H or a C₁-C₃ alkyl; orR⁶ and R⁷ when optionally taken together with the nitrogen atom to whicheach is attached form a 4 to 6 membered saturated heterocyclic ringhaving 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms, andoptionally substituted with R⁸;R⁸ is C₁-C₃ alkyl;L¹ and L⁴ are each F;L² and L³ are each H;

X is Cl;

or pharmaceutically acceptable salts thereof.

A specific embodiment of the present invention provides a method for thetreatment or prevention of tumors that express multiple drug resistance(MDR) or are resistant because of MDR in a mammal in need thereof byadministering an effective amount of a compound or pharmaceuticallyacceptable salts thereof according to Formula (II) selected from thegroup:

-   5-Azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-5-piperidin-1-yl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   7-Chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-6-[(4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl]-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   7-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,-   3-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine,-   7-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,-   N-{3-[4-(7-chloro-5-cyclohexylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propyl}-N,N-dimethylamine,-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-[(4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl]-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method for thetreatment or prevention of tumors that express multiple drug resistance(MDR) or are resistant because of MDR in a mammal in need thereof byadministering an effective amount of a compound or pharmaceuticallyacceptable salts thereof according to Formula (IIa) selected from thegroup:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

A specific embodiment of the present invention provides a method for thetreatment or prevention of tumors that express multiple drug resistance(MDR) or are resistant because of MDR in a mammal in need thereof byadministering an effective amount of a compound or pharmaceuticallyacceptable salts thereof according to Formula (IIb) selected from thegroup:

-   7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine    and-   7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.

Additionally this invention provides a method of treating, inhibitingthe growth of, or eradicating a tumor in a mammal in need thereofwherein said tumor is resistant to at least one chemotherapeutic agentwhich comprises administering to said mammal an effective amount of thecompounds of formula (II) and pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention may be prepared from: (a) commerciallyavailable starting materials (b) known starting materials which may beprepared as described in literature procedures or (c) new intermediatesdescribed in the schemes and experimental procedures herein.

Reactions are performed in a solvent appropriate to the reagents andmaterials employed and suitable for the transformation being effected.It is understood by those skilled in the art of organic synthesis thatthe various functionalities present on the molecule must be consistentwith the chemical transformations proposed. This may necessitatejudgement as to the order of synthetic steps. Appropriate considerationmust be made as to the protection of reactive functional groups toprevent undesired side reactions.

Substituents on the starting materials may be incompatible with some ofthe reaction conditions. Such restrictions to the substituents which arecompatible with the reaction conditions will be apparent to one skilledin the art. Reactions were run under inert atmospheres whereappropriate.

Compounds of Formulae (I) and (II) where R¹ is

can be prepared by a process shown in Scheme I, in which R², R³, R⁴, R⁵,and X are as hereinbefore defined. Reacting compound (III, U.S. Pat. No.6,156,925) with 2-aminoimidazole (IV) under alkaline conditions, usingtertiary amines, such as tributylamine, at a temperature up to 190° C.,provides compound (V). Halogenation with halogenating agents, POX₃ whereX is Cl or Br such as phosphorous oxychloride or phosphorous oxybromidegives 5,7-dihalo compound (VI). Replacement of the 5-chloro or 5-bromoof 5,7-dihalo compound (VI) with an excess of an amine (VII) in asuitable solvent, such as dimethylsulfoxide or dimethylformamideprovides compound of the formula (II) where R¹ is

Compounds of Formulae (I) and (II) in which R¹ is

Y represents a —O(CH₂)_(n)Q group can be alternatively prepared as shownin Scheme II by reaction of (VIII) by replacing L⁵, which is a removableleaving group, in particular a fluorine atom, with an alcohol of Formula(IX) in the presence of a strong base including alkali metal hydroxide,alkali metal carbonate and alkali hydride, e.g., sodium hydride, insuitable solvents. Suitable solvents include aprotic solvents, such asdimethylsulfoxide, dimethylformamide, and the like. The reaction issuitably carried out at a temperature in the range from about 0° C. toabout 100° C. to provide (X), a compound of formulae (I) and (II) inwhich Y represents a —O(CH₂)_(n)Q group.

Compounds of Formulae (I) and (II) in which R¹ is C₆-C₈ cycloalkyl groupcan be prepared as shown in Scheme III-VI. As described in scheme III,ester (XI) is reacted with acid chloride (XII), prepared from thecorresponding carboxylic acid where R¹ is C₆-C₈ cycloalkyl, in thepresence of lithium diisopropylamide (LDA) to give ketone (XIII) whichis further reacted with 2-aminoimidazole (IV) under alkaline conditions,using tertiary amines, such as tributylamine, at a temperature up to190° C., to give imidazo[1,2-a]pyrimidin-7-ol (XIV).

As shown in Scheme IV, imidazo[1,2-a]pyrimidin-7-ol (XIV) is reactedwith alcohol (XV) in the presence of a strong base which includes analkali metal hydroxide, alkali metal carbonate and alkali metal hydride,e.g., sodium hydride in an aprotic solvent which includesdimethylsulfoxide, dimethylformamide, and the like to give ether (XVI).Reaction of ether (XVI) with halogenating agents, POX₃ where X is Cl orBr such as phosphorous oxychloride or phosphorous oxybromide in thepresence of N,N-diethylaniline affords compound (XVII) which is furtherreacted with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to affordether (XVIII), a compound of formulae (I) and (II) where R¹ is C₆-C₈cycloalkyl, Y is —O(CH₂)_(n)Q and Q is OH.

Additionally, reaction of imidazo[1,2-a]pyrimidin-7-ol (XIV) withhalogenating agents, POX₃ where X is Cl or Br such as phosphorousoxychloride or phosphorous oxybromide affords (XIX), a compound offormula (II) where R¹ is C₆-C₈ cycloalkyl, and Y is F.

With reference to Scheme V, imidazo[1,2-a]pyrimidin-7-ol (XIV), where R¹is C₆-C₈ cycloalkyl, is reacted with amino alcohol (XX), where R⁶ and R⁷are other than H, in the presence of a strong base which includes analkali metal hydroxide, alkali metal carbonate and alkali metal hydride,e.g., sodium hydride in the presence of an aprotic solvent whichincludes: dimethylformamide, dimethyl sulfoxide, and the like to giveamine (XXI). Reaction of amine (XXI) with halogenating agents, POX₃where X is Cl or Br such as phosphorous oxychloride or phosphorousoxybromide gives ether (XXII), a compound of formulae (I) and (II) whereR¹ is C₆-C₈ cycloalkyl, Y is —O(CH₂)_(n)Q, Q is NR⁶R⁷ and R⁶ and R⁷ areother than H.

As shown in Scheme VI, imidazo[1,2-a]pyrimidin-7-ol (XIV), where R¹ isC₆-C₈ cycloalkyl, is reacted with amino alcohol (XXIII), where R⁶ is H,in the presence of a strong base which includes an alkali metalhydroxide, alkali metal carbonate and alkali metal hydride, e.g., sodiumhydride in the presence of an aprotic solvent which includes:dimethylformamide, dimethyl sulfoxide, and the like to give amine(XXIV). Protection of the nitrogen of amine (XXIV) by reaction withdi-tert-butyl dicarbonate (t-Boc)₂O affords t-Boc protected amine (XXV)which is further reacted with halogenating agents, POX₃ where X is Cl orBr such as phosphorous oxychloride or phosphorous oxybromide in thepresence of N,N-diethylaniline to afford halo compound (XXVI). Furtherreaction of halo compound (XXVI) with trifluoroacetic acid (TFA) affordsamine (XXVII), a compound of formulae (I) and (II) where R¹ is C₆-C₈cycloalkyl, Y is —O(CH₂)_(n)Q, Q is NR⁶R⁷ and R⁶ is H.

It is understood that this invention encompasses all crystalline andhydrated forms of compounds of Formulae (I) and (II) and theirpharmaceutically acceptable salts. The pharmaceutically acceptable saltsof the compounds of this invention are those derived from such organicand inorganic pharmaceutically acceptable salt forming acids as: lactic,citric, acetic, tartaric, fumaric, succinic, maleic, malonic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, benzenesulfonic, L-aspartic, R or S-mandelic, palmiticand similarly known acceptable acids. A further salt is thetrifluoroacetic acid salt (TFA). In particular the hydrochloride,fumarate and succinate salts are preferred.

The present invention accordingly provides a pharmaceutical compositionwhich comprises a compound of this invention in combination orassociation with a pharmaceutically acceptable carrier. In particular,the present invention provides a pharmaceutical composition whichcomprises an effective amount of a compound of this invention and apharmaceutically acceptable carrier. Pharmaceutical compositions of thisinvention comprise compounds of Formula (I) or Formula (II).

Based on the results of standard pharmacological test proceduresdescribed herein, the compounds of this invention are useful as agentsfor treating, inhibiting or controlling the growth of cancerous tumorcells and associated diseases in a mammal in need thereof. The compoundsof the invention are useful as agents for treating, inhibiting orcontrolling the growth of cancerous tumor cells and associated diseasesin a mammal in need thereof by interacting with tubulin and microtubulesand promoting microtubule polymerization. The compounds of the inventionare also useful for the treatment or prevention of cancerous tumors thatexpress multiple drug resistance (MDR) or are resistant because of MDR.

In particular, when contacting a tubulin containing system with aneffective amount of a compound of formulae (I) or (II) results in thepromotion of microtubule polymerization and further stabilizesmicrotubules and by promoting microtubule polymerization and stabilizingmicrotubules said compounds of formulae (I) or (II) are useful as agentsfor treating, inhibiting or controlling the growth of cancerous tumorcells and associated diseases. Additionally, compounds of formulae (I)or (II) are useful for the treatment or prevention of cancerous tumorsthat express multiple drug resistance (MDR) or are resistant because ofMDR. The tubulin containing system may be in a tumor cell, therebyinhibiting neoplastic disease by administering an effective amount of acompound described in the present invention. Mammals may be treated andin particular, humans. Further, said tubulin containing system may be ina patient. In the case of cancer treatment, it is believed that manyneoplasias such as leukemia, lung cancer, colon cancer, thyroid cancer,ovarian cancer, renal cancer, prostate cancer and breast cancers may betreated by effectively administering effective amounts of the compoundsof formulae (I) or (II). As used herein, cancer refers to all types ofcancers, or neoplasms or benign or malignant tumors. Preferred cancersfor treatment using methods provided herein include carcinoma, sarcoma,lymphoma, or leukemia. By carcinoma is meant a benign or malignantepithelial tumor and includes, but is not limited to, breast carcinoma,prostate carcinoma, non-small lung carcinoma, colon carcinoma, melanomacarcinoma, ovarian carcinoma, or renal carcinoma. A preferred host is ahuman.

The effective dosage of active ingredient employed may vary depending onthe particular compound employed, the mode of administration andseverity of the condition being treated. However, in generalsatisfactory results are obtained when the compounds of the inventionare administered in amounts ranging from about 0.10 to about 100 mg/kgof body weight per day. A preferred regimen for optimum results would befrom about 1 mg to about 20 mg/kg of body weight per day and such dosageunits are employed that a total of from about 70 mg to about 1400 mg ofthe active compound for a subject of about 70 kg of body weight areadministered in a 24 hour period.

The dosage regimen for treating mammals may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A decidedlypractical advantage is that these active compounds may be administeredin any convenient manner such as by the oral, intravenous, intramuscularor subcutaneous routes.

The active compounds of the invention may preferably be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsules, or they may be compressed into tablets or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, these active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers and the like.Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2% toabout 60% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between 10 and 1000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose, or saccharin may be added or a flavoring agnet such aspeppermint, oil of wintergreen or cherry flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose, as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amounts used.In addition, these active compounds may be incorporated intosustained-release preparations and formulations.

These active compounds may also be administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth or microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be prepared against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpoly-ethylene glycol), suitable mixtures thereof, and vegetable oils.

Intravenous administration is a preferred manner of administration ofcompounds of the invention. For intravenous administration examples ofnon-limiting suitable carriers include physiological saline,bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The composition must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. Prevention of the action of microorganismscan be achieved by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as manitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

As used in accordance with this invention, the term providing aneffective amount of a compound means either directly administering suchcompound, or administering a prodrug, derivative, or analog which willform an effective amount of the compound within the body.

In addition to the above utilities some of the compounds of thisinvention are useful for the preparation of other compounds of thisinvention.

Examples of this invention are evaluated in several standardpharmacological test procedures that showed that the compounds of thisinvention possess significant activity as promoters of microtubulepolymerization and are antineoplastic agents. Based on the activityshown in the standard pharmacological test procedures, the compounds ofthis invention are therefore useful as anticancer agents. Associatedcancers are selected from the group consisting of breast, colon, lung,prostate, melanoma, epidermal, leukemia, kidney, bladder, mouth, larynx,esophagus, stomach, ovary, pancreas, liver, skin and brain. Inparticular, the compounds of this invention possess an effect similar toPaclitaxel. The test procedures used and results obtained are shownbelow.

Standard Pharmacological Test Procedures Materials and Methods CellCulture Media and Reagents

Medium is RPMI-1640 with L-glutamine, supplemented with 10%heat-inactivated fetal calf serum, 100 units/mL penicillin, and 100μg/mL streptomycin (Gibco, Grand Island, N.Y.). Microtubule-associatedprotein (MAP)-rich tubulin, containing about 70% tubulin and 30% MAPs(#ML113), and highly purified tubulin (>99% pure, #TL238), both frombovine brain, are obtained from Cytoskeleton, Inc., Denver, Colo. PEMbuffer (80 mM piperazine-N,N′-bis[2-ethanesulfonic acid], pH 6.9, 1 mMethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mMmagnesium chloride) and guanosine 5′-triphosphate (GTP) are alsoobtained from Cytoskeleton. [³H]paclitaxel, specific activity 14.7Ci/mmol, is purchased from Moravek Biochemicals (Brea, Calif.).[³H]vinblastine, specific activity 9.60 Ci/mmol and MicroSpin G-50columns are obtained from Amersham Biosciences (Piscataway, N.J.).[³H]colchicine, specific activity 76.5 Ci/mmol, is obtained from NewEngland Nuclear (Boston, Mass.). Other reagents are obtained from Sigma(St. Louis, Mo.).

Cell Lines

Human cancer cell lines, unless otherwise noted, are obtained from theAmerican Type Culture Collection (Rockville, Md.). The followingdrug-sensitive parental cell lines, and their derived drug-resistantcounterparts, are obtained from the originators as listed: (a) S1(parental line from a subclone of human colon carcinoma line LS174T) andderived S1-M1-3.2 (herein called S1-M1) which expresses the MXR drugtransporter protein, are provided by Dr. L. Greenberger, Wyeth Research(Rabindran, S. K., He, H., Singh, M., Brown, E., Collins, K. I.,Annable, T., and Greenberger, L. M. Reversal of a novel multidrugresistance mechanism in human colon carcinoma cells by fumitremorgin C.Cancer Res., 58: 5850-5858, 1998); (b) parental HL-60 humanpromyelocytic leukemia line and derived HL-60/ADR, which expresses theMRP1 drug transporter protein, are provided by Dr. M. Center, Universityof Kansas (McGrath, T., and Center, M. S. Adriamycin resistance in HL60cells in the absence of detectable P-glycoprotein. Biochem. Biophys.Res. Commun., 145: 1171-1176, 1987), via Dr. L. Greenberger, WyethResearch; and (c) parental KB-3-1 (herein called KB, cloned from a humanepidermoid carcinoma) and the derived lines KB-8-5 and KB-V1, whichexpress moderate and very high levels of the MDR1 (P-glycoprotein) drugtransporter protein, respectively, are provided by Dr. M. Gottesman,National Cancer Institute (Shen, D. W., Cardarelli, C., Hwang, J.,Cornwell, M., Richert, N., Ishii, S., Pastan, I., and Gottesman, M. M.Multiple drug-resistant human KB carcinoma cells independently selectedfor high-level resistance to colchicine, adriamycin, or vinblastine showchanges in expression of specific proteins. J. Biol. Chem., 261:7762-7770, 1986) via Dr. L. Greenberger, Wyeth Research.

Cytotoxicity Standard Pharmacological Test Procedures

Two different standard cytotoxicity assays are used: the “MTS” assay,and the “SRB” assay.

The MTS assay, which is sold in kit form by Promega (Madison, Wis.;CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay), is basedon the conversion by viable cells, but not by dead cells, of thetetrazolium salt, MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium,inner salt), into a water-soluble colored formazan which is detected byspectrophotometry. Compounds are tested at nine concentrations, in orderto determine IC₅₀ values. For the test procedure, cells are harvested bytrypsinization (or, in the case of non-adherent cells, by simpleresuspension), washed, counted and distributed to wells of 96-wellflat-bottom microtiter plates at 1000 cells per well in 200 μL ofmedium. In addition, one row of wells on a separate plate receives cellsas above (“time 0” plate). All plates are incubated at 370 in humidified5% CO₂ in air for about 24 hr.

On day 2, compounds for test are diluted and added to wells. Compoundsare dissolved in DMSO at 10 mg/mL. For each compound, nine serial 2-folddilutions are prepared in DMSO. Ten μL of each dilution in DMSO istransferred to 100 μL of medium, mixed well, and then 5 μL of thisdilution is transferred in quadruplicate to wells containing cells. Thefinal high concentration of each compound is typically 1-5 μM. Platesare returned to the incubator for three days. At the time of drugaddition to the experimental plates, the MTS assay is run on the “time0” plate. This gives the “time 0 MTS value” which is related to thenumber of viable cells per well at the time of drug addition.

After three days of culture with test compounds (day 5 overall), the MTSassay is done on all wells of the experimental plates. The absorbancevalues of the quadruplicate sample wells are averaged and divided by theaverage of the “time 0” values. The average of control wells withoutdrug, divided by the average “time 0” value, gives the maximal relativeincrease in MTS color yield due to cell growth during the final threedays of culture. The average of control wells with high drugconcentration, divided by the “time 0” value, gives the minimal relativecolor yield for cells that were completely killed. The nine values foreach compound are plotted against concentration, and the concentrationthat produces a relative color yield half way between the maximum andminimum is taken as the IC₅₀ value. The most potent compounds have thelowest IC₅₀ values.

The SRB standard cytotoxicity assay is done according to previouslyreported methods (Discafani, C. M., Carroll, M. L., Floyd Jr., M. B. F.,Hollander, I. J., Husain, Z., Johnson, B. D., Kitchen, D., May, M. K.,Malo, M. S., Minnick Jr., A. A., Nilakantan, R., Shen, R., Wang Y-F.,Wissner, A., Greenberger, L. M. Irreversible inhibition of epidermalgrowth factor receptor tyrosine kinase with in vivo activity byN-[4-[3-bromophenyl)amino]-6-quinaxolinyl]-2-butynamide (CL-387,785).Biochem. Pharmacol., 57:917-925, 1999). Briefly, cells are plated in 100μL of medium in 96-well flat-bottom microtiter plates in the morning ofday 1 and allowed to adhere to the plates for 2-6 hr. Compounds areserially diluted into medium as 2× stocks and 100 μL of these stocks areadded to cells in duplicate. Compounds are incubated with cells for 3days. At the end of the incubation period the sulforhodamine B (SRB)assay, which measures protein content as an assessment of cell survival,is performed as described previously (Skehan, P., Storeng, R., Scudiero,D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H.,Kenney, S., Boyd, M. R. New calorimetric cytotoxicity assay foranticancer-drug screening. J. Natl. Cancer Inst., 82: 1107-1112, 1990)with some modifications. Medium is gently decanted from plates andreplaced with 200 μL per well of cold serum-free medium containing afinal concentration of 10% trichloroacetic acid. The plates areincubated for 1 hr at 4°, washed 5 times in cold distilled water, andair-dried overnight. The fixed cells are stained for 10 min with 80 μLper well of 0.04% SRB solution prepared in 1% glacial acetic acid.Staining solution is decanted, plates are washed 5 times in 1% glacialacetic acid, and then air-dried until completely dry. Stained cellproduct is dissolved in 150 μL per well of 10 mM Trizma Base withshaking for 20 minutes. Absorbance is read on a Victor V multi-labelplate reader (Perkin Elmer, Gaithersburg, Md.).

Tubulin Polymerization Standard Pharmacological Test Procedure

Two variations of this procedure are done, one using MAP-rich tubulinand one using pure tubulin.

MAP-rich tubulin is dissolved in ice-cold PEM buffer containing 1 mM GTP(GPEM buffer) at a concentration of 1.3 mg/mL. The solution iscentrifuged at top speed in an Eppendorf model 5415C microcentrifuge(Brinkmann Instruments, Westbury, N.Y.) for 10 min at 40 immediatelybefore use. The tubulin solution is added to wells of a ½-area 96-wellplate (Costar No. 3696, Corning, Inc., Corning, N.Y.) already containingthe compounds of interest. Each compound is tested in duplicate at afinal concentration of 0.3 μM in a volume of 110 μL per well. The finalDMSO concentration in all wells is 0.3%. Control reactions, whichreceive compound solvent only, are done in quadruplicate. The plate isput in a SpectraMax Plus plate reader (Molecular Devices Corp.Sunnyvale, Calif.) thermostated at 24° and the absorbance of each wellat 340 nm, a measure of the appearance of turbidity due to tubulinpolymer formation, is determined every minute for 60 minutes. Theabsorbance at time 0 for each well is subtracted from each of thesubsequent absorbance readings for that well, and then the duplicatesare averaged.

The procedure with pure tubulin is similar except for the followingchanges. Pure tubulin is dissolved in cold PEM buffer containing 10%glycerol and no added GTP at a concentration of 1.5 to 1.8 mg/mL (15 to18 μM). The supernatant after centrifugation is dispensed to a 1/2-area96-well plate already containing compounds. Each compound is tested induplicate at a final concentration of 24.3 μM. The plate reader isthermostated at 350.

Competitive Binding Standard Pharmacological Test Procedure

The binding of examples of this invention to highly purified tubulin isstudied by competitive inhibition methods. The αβ-tubulin heterodimercontains binding sites for the three major classes of microtubule-activepharmacological agents: taxanes, vinca/peptide-site agents, andcolchicine-site agents. To study possible competition at thevinca/peptide and colchicine sites, incubations are done underconditions which do not favor polymerization because vinblastine andcolchicine bind preferentially to unpolymerized heterodimer. To studypossible competition at the taxane site, on the other hand, polymerizedtubulin (microtubules) is used because paclitaxel binds preferentiallyto microtubules.

Highly purified tubulin is dissolved in PEM buffer without GTP and usedat a final concentration of 1.0 to 1.3 mg/ml (10 to 13 μM). To aliquotsof the tubulin solution are added various competitors (inquadruplicates) at 100 μM final concentrations, and [³H]vinblastine or[³H]colchicine at final concentrations of 100 nM or 50 nM, respectively.These solutions are incubated at 240 for 1 hr and then applied toMicroSpin G-50 columns which are centrifuged for 2 min at 3000 rpm in anEppendorf 5415C microfuge. An aliquot of each column effluent(containing tubulin and bound radioligand) is mixed with scintillationfluid and counted in a liquid scintillation spectrometer. Controlsinclude samples without competitor, and samples with unlabeledvincristine, colchicine, or paclitaxel. Quadruplicates are averaged, andthe ability of the competitor to inhibit the binding of the radioligandis expressed as a percentage of control binding in the absence of anycompetitor.

For competition with [³H]paclitaxel, highly purified tubulin isdissolved in PEM buffer containing 0.75 M glutamate and 25 μMdideoxy-GTP; final protein concentration is 0.25 to 0.35 mg/mL (2.5 to3.5 μM). These conditions foster the rapid formation of short, stablemicrotubule polymers (Hamel, E., del Campo, A. A., and Lin, C. M.Stability of tubulin polymers formed with dideoxyguanosine nucleotidesin the presence and absence of microtubule-associated proteins. J. Biol.Chem., 259: 2501-2508, 1984). This solution is incubated for 30 min at37° to allow microtubules to form. Then [³H]paclitaxel (finalconcentration of 2.1 μM, 1.2 Ci/mmol) and competitor (finalconcentration of 20 μM, except 5 μM for unlabeled paclitaxel) are addedto aliquots of the polymerized tubulin solution and incubation at 370 iscontinued for another 30 min. Controls include samples withoutcompetitor, and samples with unlabeled vincristine, colchicine, orpaclitaxel. The reactions are then centrifuged at top speed in anEppendorf 5415C microfuge for 20 min at room temperature in order topellet the microtubule protein. Triplicate aliquots of each supernatantare mixed with scintillation fluid and counted in a liquid scintillationspectrometer. From the amount of radioactivity in the supernatants andthe measured total starting radioactivity, the amount of [³H]paclitaxelbound to pelleted microtubule protein is calculated. The ability of eachcompetitor to inhibit radioligand binding to pelleted protein isexpressed as a percent of controls without any competitor.

Results 1. Cytotoxicity Standard Pharmacological Test Procedure

1.1. With COLO 205 Cells

COLO 205 is a human colon carcinoma cell line that is used forcomparative testing of the examples of this invention and severalreference compounds (Table 1). This line is sensitive to paclitaxel andvincristine.

TABLE 1 Activity of Examples of the Invention and Reference Compounds inthe MTS Cytotoxicity Standard Pharmacological Test Procedure with COLO205 Cells¹ Example or Reference IC₅₀ Compound (nM) SD n 1 259 74 3 2 3968 2 3 742 385 2 4 88 26 2 5 207 111 2 6 45 17 3 Paclitaxel 3.3 1.0 20Vincristine 2.6 0.5 7 ¹IC₅₀ values and standard deviations are from theindicated number of independent experiments

1.2. With KB, KB-8-5, and KB-V1 Cells

The KB lines express different amounts of the P-glycoprotein (MDR1)membrane pump which produces resistance to the action of many cytotoxiccompounds, including paclitaxel and vincristine. The parental KB lineexpresses no P-glycoprotein, KB-8-5 expresses moderate levels of theprotein, and KB-V1 expresses very high levels. The ability ofP-glycoprotein to recognize and export a potential cytotoxic agent canbe inferred from the change in IC₅₀ values on these lines (Loganzo, F.,Discafani, C. M., Annable, T., Beyer, C., Musto, S., Hari, M., Tan, X.,Hardy, C., Hernandez, R., Baxter, M., Singanallore, T., Khafizova, G.,Poruchynsky, M. S., Fojo, T., Nieman, J. A., Ayral-Kaloustian, S., Zask,A., Andersen, R. J., and Greenberger, L. M. HTI-286, a syntheticanalogue of the tripeptide hemiasterlin, is a potent antimicrotubuleagent that circumvents P-glycoprotein-mediated resistance in-vitro andin vivo. Cancer Res., 63:1838-1845, 2003). If a compound is recognizedby P-glycoprotein, its IC₅₀ value will increase substantially (severalhundred-fold) on going from KB to KB-8-5 to KB-V1; if a compound is notrecognized, it will have similar IC₅₀ values (3-fold or less difference)on all three lines. For example, as shown in Table 2, KB-8-5 cells aremoderately resistant to paclitaxel (19-fold), vincristine (11-fold),colchicine (3.4-fold) and doxorubicin (3.0-fold). In contrast, example 6of this invention shows a 1.4-fold change in IC₅₀ values. In addition,example 4, tested in the SRB cytotoxicity assay procedure (Table 3), hasa ratio of 2.5 compared with a ratio of 16 for paclitaxel.

Even slight interactions of compounds with P-glycoprotein can bedetermined with the KB-V1 line, which expresses a level of this proteinhigher than is typically found in clinical samples from a variety oftumors (Goldstein, L. J., Galski, H., Fojo, T., Willingham, M., Lai, S.L., Gazdar, A., Pirker, R., Green, A., Crist, W., Brodeur, G. M.,Lieber, M., Cossman, J., Gottesman, M. M., and Pastan, I. Expression ofa multidrug resistance gene in human cells. J. Natl. Cancer Inst.(Bethesda), 81: 116-124, 1989). KB-V1 cells are highly resistant topaclitaxel (>345-fold), vincristine (>156-fold), colchicine (116-fold),mitoxantrone (77-fold), and doxorubicin (>130-fold). Example 6 of thisinvention shows a 4.6-fold change in IC₅₀ compared to the parental KBline (Table 2). This indicates that example 6 is barely recognized byP-glycoprotein and therefore that it overcomes P-glycoprotein-mediatedresistance to cell killing. Example 4, tested in the SRB cytotoxicityassay procedure (Table 3), has a ratio of 99 compared with a ratioof >1,360 for paclitaxel in the same assay, indicating that althoughexample 4 shows greater recognition by P-glycoprotein than example 6, ittoo is recognized much less than paclitaxel.

TABLE 2 Activity of Example 6 of the Invention and Reference Compoundsin the MTS Cytotoxicity Standard Pharmacological Test Procedure with KB,KB-8.5 and KB-VI Cells Example or Reference IC₅₀ (nM)¹ Ratio² CompoundKB KB 8.5 KB VI 8.5/KB VI/KB 6 44 63 204 1.4 4.6 Paclitaxel 2.956 >1,000 19 >345 Vincristine 6.4 72 >1,000 11 >156 Colchicine 18 592,038 3.4 116 Camptothecin 24 33 39 1.4 1.6 Mitoxantrone 25 27 1,927 1.177 Doxorubicin 23 70 >3,000 3.0 >130 ¹IC₅₀ values are means of 2independent experiments. ²Ratio = IC₅₀ on KB 8.5 or KB VI cells/IC₅₀ onKB cells. A ratio of about 1 indicates no resistance.

TABLE 3 Activity of Example 4 of the Invention and Paclitaxel in the SRBCytotoxicity Standard Pharmacological Test Procedure with KB, KB-8.5 andKB-VI Cells Example or Reference IC₅₀ (nM)¹ Ratio² Compound KB KB 8.5 KBVI 8.5/KB VI/KB 4 24 59 2387 2.5 99 Paclitaxel 2.2 34 >3000 16 >1360¹IC₅₀ values are means of 2 independent experiments. ²Ratio = IC₅₀ on KB8.5 or KB VI cells/IC₅₀ on KB cells. A ratio of about 1 indicates noresistance.

1.3. With HL-60 and HL-60/ADR Cells

HL-60/ADR cells overexpress the multidrug resistance protein MRP1 whichmediates resistance to some chemotherapeutics (Gottesman, M. M., Fojo,T., and Bates, S. E. Multidrug resistance in cancer: role ofATP-dependent transporters. Nature Rev. Cancer, 2: 48-58, 2002). TheIC₅₀ values of example 6 of this invention, as well as referencecompounds, on HL-60/ADR are compared to values on the sensitive parentalHL-60 line. The results, shown in Table 4, indicate that whereasHL-60/ADR cells show resistance to vincristine (8.2-fold), colchicine(7.4-fold), mitoxantrone (17-fold), and doxorubicin (93-fold), thesecells show no resistance to example 6. This indicates that example 6 isnot recognized by MRP1 and therefore overcomes cellular resistancemediated by this transporter.

TABLE 4 Activity of Example 6 of the Invention and Reference Compoundsin the MTS Cytotoxicity Standard Pharmacological Test Procedure withHL-60 and HL-60/ADR Cells Example or Reference IC₅₀ (nM)¹ Compound HL-60HL-60/ADR Ratio² 6 53 29 0.55 Paclitaxel 5.7 6.4 1.1 Vincristine 2.5 208.2 Colchicine 9.3 69 7.4 Camptothecin 12 17 1.4 Mitoxantrone 9.5 161 17Doxorubicin 23 2,085 93 ¹IC₅₀ values are means of 2 independentexperiments. ²Ratio = IC₅₀ on HL-60/ADR cells/IC₅₀ on HL-60 cells. Aratio of about 1 indicates no resistance.

1.4. With S1 and S1-M1 Cells

S1-M1 cells overexpress the MXR transporter which mediates resistance tosome chemotherapeutics (Gottesman, M. M., Fojo, T., and Bates, S. E.Miltidrug resistance in cancer: role of ATP-dependent transporters.Nature Rev. Cancer, 2: 48-58, 2002). The IC₅₀ values of example 6 ofthis invention, as well as reference compounds, on S1-M1 are compared tovalues on the sensitive parental S1 line. The results, shown in Table 5,indicate that whereas S1-M1 cells show resistance to mitoxantrone(>300-fold) and doxorubicin (74-fold), they show no resistance toexample 6. This indicates that example 6 is not recognized by MXR andtherefore overcomes cellular resistance mediated by this transporter.

TABLE 5 Activity of Example 6 of the Invention and Reference Compoundsin the MTS Cytotoxicity Standard Pharmacological Test Procedure with S1and S1-M1 Cells Example or Reference IC₅₀ (nM)¹ Compound S1 S1-M1 Ratio²6 63 68 1.1 Paclitaxel 8.1 4.4 0.54 Vincristine 5.6 4.6 0.82 Colchicine18 60 3.3 Camptothecin 8.9 17 1.9 Mitoxantrone 10 >3,000 >300Doxorubicin 34 2,517 74 ¹IC₅₀ values are means of 2 independentexperiments. ²Ratio = IC₅₀ on S1-M1 cells/IC₅₀ on S1 cells. A ratio ofabout 1 indicates no resistance.

2. Effects of Compounds on Polymerization of MAP-Rich and Pure TubulinIn Vitro

In this assay, control reactions with MAP-rich tubulin show an S-shapedabsorbance profile characterized by three phases: first, a lag phaseduring which no change in absorbance occurs; second, a polymerizationphase in which absorbance increases; and third, a plateau phase in whichabsorbance has reached a maximum and little or no further change occurs.Polymerization enhancers such as paclitaxel and docetaxel shorten oreliminate the lag phase, increase the rate of the polymerization phase,and often increase the height of the plateau. Polymerization inhibitorssuch as vincristine and colchicine reduce or prevent the absorbanceincrease. Example 6 of this invention has a taxane-like effect on thepolymerization reaction. This has been expressed quantitatively in Table6 by dividing the mean A₃₄₀ of each sample at 20 min by the mean A₃₄₀ ofthe control at 20 min to give a fold enhancement over control.Paclitaxel and docetaxel show enhancement factors of 1.8 and 5.4,respectively. Example 6 of this invention has an enhancement factor of1.7. In contrast, vincristine has an enhancement factor of 0.5 becauseit partially inhibits polymerization of MAP-rich tubulin.

TABLE 6 Activity of Example 6 of the Invention and Reference Compoundsin the Tubulin Polymerization Standard Pharmacological Test Procedurewith MAP-rich Tubulin Example or Reference A₃₄₀ Compound Compound A₃₄₀Control 6 1.7 Paclitaxel 1.8 Docetaxel 5.4 Vincristine 0.5 Control 1.0

Pure tubulin without added GTP shows no polymerization in controlreactions. Docetaxel and paclitaxel are able to induce polymerization ofpure tubulin under these conditions. Example 6 of this invention alsoinduces polymerization of pure tubulin without GTP in a manner similarto docetaxel. Table 7 shows the mean absorbance at four time pointsafter the start of the reactions for a single compound concentration. Atthis concentration (24.3 μM) docetaxel and example 6 cause a rapidincrease in absorbance within the first 5 min of reaction to a plateau.The microtubule destabilizers vincristine and colchicine show noactivity in this assay.

TABLE 7 Activity of Example 6 of the Invention and Reference Compoundsin the Tubulin Polymerization Standard Pharmacological Test Procedurewith Pure Tubulin Example or Reference A₃₄₀ at Compound 0 min 5 min 10min 15 min 20 min 6 0 0.15 0.19 0.19 0.19 Docetaxel 0 0.20 0.20 0.200.20 Vincristine 0 0.01 0.01 0 0 Colchicine 0 0 0 0 0 Control 0 0 0 0 0

Binding of Compounds to Tubulin

The site on highly purified bovine brain tubulin to which compounds ofthis invention bind is determined by competitive inhibition studies withthe radioactive ligands [³H]vinblastine, [³H]colchicine, and[³H]paclitaxel. The results, shown in Table 8, indicate that example 6inhibits the binding of [³H]vinblastine to tubulin heterodimer (17% ofcontrol), but does not inhibit binding of [³H]colchicine to tubulinheterodimer or of [³H]paclitaxel to microtubules. This is strongevidence that this example binds at the vinca/peptide site of tubulinand not at the colchicine or taxane sites. Among the control compoundstested, vincristine inhibited [³H]vinblastine binding but not[³H]colchicine, and colchicine inhibited [³H]colchicine binding but not[³H]vinblastine. Vincristine and colchicine also appear to inhibit thebinding of [³H]paclitaxel to microtubules; however, this is not due tobinding competition but rather to depolymerization of the microtubulesto which [³H]paclitaxel binds. It is clear that example 6 of thisinvention does not reduce [³H]paclitaxel binding to microtubules, whichindicates that it neither competes with [³H]paclitaxel for binding nordepolymerizes the microtubules to which [³H]paclitaxel binds.

TABLE 8 Activity of Example 6 of the Invention and Reference Compoundsin the Competitive Binding Standard Pharmacological Test Procedure¹Radioactive Ligand [³H]Vinblastine [³H]Colchicine [³H]PaclitaxelCompetitor Mean² SD² Mean² SD² Mean³ SD³ Control 100 100 100 Example 617 1.7 84 3.5 92 6.5 Vincristine 5 1.0 99 7.9 22 0.9 Colchicine 125 12.66 1.9 19 0.2 Paclitaxel 92 7.8 93 12.3 35 1.6 ¹Results are expressed aspercent of binding to control without competitor. ²Data are from 1 (4replicates) or 2 (8 replicates) independent experiments. ³Data are from1 to 4 independent experiments (3 to 12 replicates).

Compounds of this invention show potent cytotoxic activity againstmultiple human cancer cell lines in culture, including lines that areresistant to paclitaxel and vincristine because of drug transporteroverexpression. The compounds enhance the initial rate of MAP-richtubulin polymerization, in a manner reminiscent of taxanes and distinctfrom the inhibitory effects of depolymerizers such as vinca alkaloidsand colchicine. They also induce polymerization of pure tubulin in theabsence of GTP. Compounds of this invention bind to the vinca/peptidesite of tubulin.

The following examples further illustrate the present invention. Itshould be understood, however, that the invention is not limited solelyto the particular examples given below.

EXAMPLE 1 Preparation of5-azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine

Step A: 5,7-dichloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine

A mixture of diethyl 2-(2,4,6-trifluorophenyl)malonate (U.S. Pat. No.6,156,925) (870 mg, 3.0 mmol), 2-aminoimidazole (Hel. Acta. Chim. 76,2066 (1993))(274 mg, 3.3 mmol), and 1.0 mL of tributylamine is stirredunder nitrogen atmosphere at 160° C. for 0.5 h and cooled to roomtemperature. The mixture is dissolved in ethyl acetate and the organiclayer is washed with 1.0 N hydrochloric acide (x3) and saturated sodiumchloride, dried over magnesium sulfate, and concentrated. The residue isdissolved in ethyl acetate (5 mL) and to this solution is added hexanes(50 mL). The precipitates are collected by filtration, washed withhexanes to give6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine-5,7-diol as a tansolid (180 mg). MS: m/z 279.9(M−H).

A mixture of 6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine-5,7-diol(180 mg) in 1 mL of phosphorous oxychloride is heated at reflux for 6 h.The excess phosphorous oxychloride is removed in vaccuo, and theresulting residue is dissolved in methylene chloride. The organic layeris washed with water, dried over magnesium sulfate, and concentrated.The residue is chromatographed over silica gel, eluting with a gradientof 10% ethyl acetate in hexanes to 33% ethyl acetate in hexanes.Concentration provides5,7-dichloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine as awhite solid (62 mg). MS: m/z 318.0 (M+H).

Step B:5-Azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine

A solution of5,7-dichloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine (16 mg,0.05 mmol) and hexamethyleneimine (100 mg, 1.0 mmol) in 1 mL ofmethylene chloride is stirred at room temperature for 16 h. The organicsolution is washed with 0.1 N hydrochloric acid and saturated sodiumchloride, dried over magnesium sulfate, and concentrated. The residue ischromatographed over silica gel, eluting with a gradient of 20% ethylacetate in hexanes to 50% ethyl acetate in hexanes. Concentrationprovides5-azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidineas a yellow solid (15 mg, mp 106-108° C.). MS: m/z 381.0 (M+H).

Example 2 is synthesized analogously to Example 1.

EXAMPLE 27-Chloro-5-piperidin-1-yl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine;MS: m/z 367.3(M+H)

EXAMPLE 3 Preparation of7-chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine;

A solution of5,7-dichloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine (16 mg,0.05 mmol) and 2,2,2-trifluoroethylamine (200 mg, 2.0 mmol) in 1 mL ofN,N-dimethylformamide is stirred at room temperature for 16 h. Asaturated sodium chloride solution is added, and the product isextracted with ethyl acetate. The organic solution is washed withsaturated sodium chloride, dried over magnesium sulfate, andconcentrated. The residue is chromatographed over silica gel, elutingwith a gradient of 20% ethyl acetate in hexanes to 50% ethyl acetate inhexanes. Concentration provides7-chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amineas a white solid (16 mg, mp 155-157° C.). MS: m/z 381.0 (M+H).

EXAMPLE 4 Preparation7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine;

To a solution of7-chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine(19 mg, 0.05 mmol) and 3-dimethylamino-1-propanol (51 m g, 0.5 mmol) in0.5 mL of dimethylsulfoxide at room temperature is added sodium hydride(60% in mineral oil, 20 mg, 0.5 mmol). The mixture is heated at 50° C.for 30 minutes, and cooled to room temperature. A saturated sodiumchloride solution is added, and the product is extracted with ethylacetate. The organic solution is washed with saturated sodium chloride(x3), dried over magnesium sulfate, and concentrated. The residue ischromatographed over silica gel, eluting with a gradient of ethylacetate to 30% methyl alcohol in ethyl acetate. Concentration provides7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amineas a tan solid (12 mg, mp 52-55° C.). MS: m/z 464.3 (M+H).

EXAMPLE 57-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine;

Step A: Ethyl 3-cycloheptyl-3-oxo-2-(2,4,6-trifluorophenyl)propanoate

A mixture of 2,4,6-trifluorophenylacetic acid (570 mg, 3.0 mmol),iodoethane (1.56 g, 10 mmol), and potassium carbonate (1.38 g, 10 mmol)in 5 mL of dimethylsulfoxide is stirred at 50° C. for 3 h, and cooled toroom temperature. The mixture is partitioned between diethyl ether andwater. The organic layer is washed with water, and saturated sodiumchloride, dried over magnesium sulfate, and filtered through magnesol.The filtrate is concentrated to give ethyl 2,4,6-trifluorophenylacetateas a light yellow oil (581 mg, 2.66 mmol).

A mixture of cycloheptanecarboxylic acid (5.0 g, 35.2 mmol) in 25 mL ofthionyl chloride is refluxed for 1 h, and concentrated. The crudecycloheptanecarboxylic acid chloride thus obtained is used directly inthe next step.

A solution of ethyl trifluorophenylacetate (436 mg, 2.0 mmol) in 3 mL oftetrahydrofuran is cooled to −78° C., and lithium diisopropylamide (2.0M in heptane/tetrahydrofuran/ethylbenzene, 1.0 mL, 2.0 mmol) is addeddropwise with stirring. The mixture is stirred at −78° C. for 1 h, andcycloheptanecarboxylic acid chloride (321 mg, 2.0 mmol) is addeddropwise. The mixture is warmed to room temperature and acidified with 2mL of 1.0 N hydrochloric acid. The product is extracted with ethylacetate. The organic layer is washed with saturated sodium chloride,dried over magnesium sulfate, and concentrated. The residue ischromatographed over silica gel, eluting with a gradient of hexanes to10% ethyl acetate in hexanes. Concentration provides ethyl3-cycloheptyl-3-oxo-2-(2,4,6-trifluorophenyl)propanoate as a colorlessoil (410 mg). MS: m/z 341.2 (M−H).

Step B:7-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine;

A mixture of ethyl3-cycloheptyl-3-oxo-2-(2,4,6-trifluorophenyl)propanoate (342 mg, 1.0mmol), 2-aminoimidazole (Helv. Acta. Chim. 76, 2066 (1993))(83 mg, 1.0mmol), and 0.5 mL of tributylamine is stirred under nitrogen atmosphereat 160° C. for 1.5 h and cooled to room temperature. The mixture isdissolved in ethyl acetate and the organic layer is washed with 1.0 Nhydrochloric acide (x2) and saturated sodium chloride, dried overmagnesium sulfate, and concentrated to give crude5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-7-ol as adark oil (294 mg).

A mixture of the above5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-7-ol (294mg) in 2 mL of phosphorous oxychloride is heated at reflux for 6 h. Theexcess phosphorous oxychloride is removed in vaccuo, and the resultingresidue is dissolved in methylene chloride. The organic layer is washedwith water, dried over magnesium sulfate, and concentrated. The residueis chromatographed over silica gel, eluting with a gradient of 10% ethylacetate in hexanes to 33% ethyl acetate in hexanes. Concentrationprovides7-chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidineas a white solid (24 mg). MS: m/z 380.2 (M+H).

EXAMPLE 63-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine;

A mixture of ethyl3-cycloheptyl-3-oxo-2-(2,4,6-trifluorophenyl)propanoate (342 mg, 1.0mmol), 2-aminoimidazole (Hel. Acta. Chim. 76, 2066 (1993))(83 mg, 1.0mmol), and 0.5 mL of tributylamine is stirred under nitrogen atmosphereat 160° C. for 1.5 h and cooled to room temperature. The mixture isdissolved in ethyl acetate and the organic layer is washed with 1.0 Nhydrochloric acide (x2) and saturated sodium chloride, dried overmagnesium sulfate, and concentrated to give crude5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-7-ol as adark oil.

To a solution of the above5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-7-ol and3-dimethylamino-1-propanol (206 m g, 2.0 mmol) in 3.0 mL ofdimethylsulfoxide at room temperature is added sodium hydride (60% inmineral oil, 80 mg, 2.0 mmol). The mixture is heated at 40° C. for 2 h,and cooled to room temperature. A saturated sodium chloride solution isadded, and the product is extracted with ethyl acetate. The organicsolution is washed with saturated sodium chloride (x3), dried overmagnesium sulfate, and concentrated. To the residue is added 5 mL ofphosphorous oxychloride and 2 mL of N,N-diethylaniline, and the mixtureis heated at 110° C. for 1 h. The excess phosphorous oxychloride isremoved in vaccuo, and the resulting residue is partitioned betweenethyl acetate and 5% sodium carbonate solution. The organic layer iswashed with saturated sodium chloride, dried over magnesium sulfate, andconcentrated. The residue is chromatographed over silica gel, elutingwith a gradient of ethyl acetate to 20% methyl alcohol in ethyl acetate.Concentration provides3-[4-(7-chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amineas a brown oil (24 mg). MS: m/z 463.3 (M+H).

Diethyl 2-(2,4,6-trifluorophenyl)malonate is disclosed in U.S. Pat. No.6,156,925. 2-Aminoimidazole is prepared as described in Helv. Acta.Chim. 76, 2066 (1993).

1-18. (canceled)
 19. A method of treating or inhibiting the growth ofcancerous tumor cells and associated diseases in a mammal in needthereof which comprises administering an effective amount of a compoundof formula (II).

wherein: R¹ is selected from

and C₆-C₈ cycloalkyl; R² is a moiety of the formula

R³ is H, or C₁-C₃ alkyl; R⁴ is H, or C₁-C₃ alkyl; or R³ and R⁴optionally taken together form a 6 to 8 membered saturated heterocyclicring having 1-2 nitrogen atoms, 0-1 oxygen atoms and 0-1 sulfur atoms,and optionally substituted with C₁-C₃ alkyl; R⁵ is H, C₁-C₃ alkyl orC₁-C₃ fluoroalkyl; Y is H, F, Cl, or a moiety of the formula—O(CH₂)_(n)Q; n is an integer of 2, 3 or 4; Q is —OH, or —NR⁶R⁷; R⁶ andR⁷ are independently H or C₁-C₃ alkyl; or R⁶ and R⁷ when optionallytaken together with the nitrogen atom to which each is attached form a 4to 6 membered saturated heterocyclic ring having 1-2 nitrogen atoms, 0-1oxygen atoms and 0-1 sulfur atoms, and optionally substituted with R⁸;R⁸ is C₁-C₃ alkyl; L¹, L², L³, and L⁴ are each independently H, F, Cl,Br or CF₃; X is Cl or Br; or pharmaceutically acceptable salts thereof.20. A method according to claim 19 wherein R² is a moiety of the formula

or pharmaceutically acceptable salts thereof is administered.
 21. Amethod according to claim 19 wherein Formula (II) is represented byFormula (IIa):

or pharmaceutically acceptable salts thereof is administered.
 22. Amethod according to claim 19 wherein R² is the moiety

R³ is H, or C₁-C₃ alkyl; R⁴ is H; R⁵ is C₁-C₃ fluoroalkyl; Y is F, or amoiety —O(CH₂)_(n)Q; n is 3; Q is —NR⁶R⁷; R⁶ and R⁷ are eachindependently H or a C₁-C₃ alkyl; or R⁶ and R⁷ when optionally takentogether with the nitrogen atom to which each is attached form a 4 to 6membered saturated heterocyclic ring having 1-2 nitrogen atoms, 0-1oxygen atoms and 0-1 sulfur atoms, and optionally substituted with R⁸;R⁸ is C₁-C₃ alkyl; L¹ and L⁴ are each F; L² and L³ are each H; X is Cl;or pharmaceutically acceptable salts thereof is administered.
 23. Amethod according to claim 19 wherein R¹ is a is C₆-C₈ cycloalkyl orpharmaceutically acceptable salts thereof is administered.
 24. A methodaccording to claim 19 wherein R¹ is C₆-C₈ cycloalkyl; R² is the moiety

Y is F, or a moiety —O(CH₂)_(n)Q; n is 3; Q is —NR⁶R⁷; R⁶ and R⁷ areeach independently H or a C₁-C₃ alkyl; or R⁶ and R⁷ when optionallytaken together with the nitrogen atom to which each is attached form a 4to 6 membered saturated heterocyclic ring having 1-2 nitrogen atoms, 0-1oxygen atoms and 0-1 sulfur atoms, and optionally substituted with R⁸;R⁸ is C₁-C₃ alkyl; L¹ and L⁴ are each F; L² and L³ are each H; X is Cl;or pharmaceutically acceptable salts thereof is administered.
 25. Amethod according to claim 21 wherein R² is the moiety

R³ is H, or C₁-C₃ alkyl; R⁴ is H; R⁵ is C₁-C₃ fluoroalkyl; Y is F, or amoiety —O(CH₂)_(n)Q; n is 3; Q is —NR⁶R⁷; R⁶ and R⁷ are eachindependently H or a C₁-C₃ alkyl; or R⁶ and R⁷ when optionally takentogether with the nitrogen atom to which each is attached form a 4 to 6membered saturated heterocyclic ring having 1-2 nitrogen atoms, 0-1oxygen atoms and 0-1 sulfur atoms, and optionally substituted with R⁸;R⁸ is C₁-C₃ alkyl; L¹ and L⁴ are each F; L² and L³ are each H; X is Cl;or pharmaceutically acceptable salts thereof is administered.
 26. Amethod according to claim 19 wherein Formula (II) is represented byFormula (IIb):

or pharmaceutically acceptable salts thereof is administered.
 27. Amethod according to claim 26 wherein R² is the moiety

R³ is H, or C₁-C₃ alkyl; R⁴ is H; R⁵ is C₁-C₃ fluoroalkyl; Y is F, or amoiety —O(CH₂)_(n)Q; n is 3; Q is —NR⁶R⁷; R⁶ and R⁷ are eachindependently H or a C₁-C₃ alkyl; or R⁶ and R⁷ when optionally takentogether with the nitrogen atom to which each is attached form a 4 to 6membered saturated heterocyclic ring having 1-2 nitrogen atoms, 0-1oxygen atoms and 0-1 sulfur atoms, and optionally substituted with R⁸;R⁸ is C₁-C₃ alkyl; L¹ and L⁴ are each F; L² and L³ are each H; X is Cl;or pharmaceutically acceptable salts thereof is administered.
 28. Amethod according to claim 19 wherein the compound is selected from thegroup:5-Azepan-1-yl-7-chloro-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,7-Chloro-5-piperidin-1-yl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,7-Chloro-N-(2,2,2-trifluoroethyl)-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidin-5-amine,7-Chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,7-Chloro-5-cycloheptyl-6-(2,4,6-trifluorophenyl)imidazo[1,2-a]pyrimidine,3-[4-(7-Chloro-5-cycloheptylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]-N,N-dimethylpropan-1-amine,7-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-N-(2,2,2-trifluoroethyl)imidazo[1,2-a]pyrimidin-5-amine,N-{3-[4-(7-chloro-5-cyclohexylimidazo[1,2-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propyl}-N,N-dimethylamine,7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amineand7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-(−2,2,2-trifluoro-1-methylethyl)imidazo[1,2-a]pyrimidin-5-amineor a pharmaceutically acceptable salt thereof is administered.
 29. Amethod according to claim 21 wherein the compound is selected from thegroup:7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amineand7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amine.or a pharmaceutically acceptable salt thereof is administered.
 30. Amethod according to claim 26 wherein the compound is selected from thegroup:7-chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amineand7-chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophenyl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]imidazo[1,2-a]pyrimidin-5-amineor a pharmaceutically acceptable salt thereof is administered. 31-80.(canceled)